Project Summary/Abstract The goals of these proposed studies are to establish how naturally occurring sequence heterogeneity and capping at the 5?-end of the HIV-1 genomic RNA influences its structure and function and to determine the three-dimensional structure of the 5? leader RNA in its monomeric conformation using nuclear magnetic resonance. All lentiviral genomes are transcribed in infected cells from an integrated proviral DNA that contains a stretch of three sequential guanosines, any of which could potentially serve as the transcription start site. Recently, Kawai and co-workers reported that cells infected with HIV-1 produce both 5-capped HIV-1 genomes that begin with one, two, or three guanosines (1G, 2G, and 3G respectively), that the 1G genome is specifically packaged into virions, and that 5-end heterogeneity influences reverse transcription. In collaboration with Dr. Telesnitsky at the University of Michigan, we have obtained similar evidence that HIV-1 5-end heterogeneity influences RNA fate. The 5-capped 1G genomes were selected efficiently for packaging and the 2G and 3G genomes were enriched on polysomes, apparently preferred for translation and possibly for splicing. In this proposal, I present evidence that 5-end heterogeneity significantly influences the structure of the RNA in vitro. Native gel electrophoresis studies reveal that 5-capped 1G leader RNAs readily form dimers, whereas 5-capped 2G and 3G RNAs adopt stable monomers. I hypothesize that the additional guanosine enables the 5-cap to disrupt base pairing in the lower stem of the adjacent polyA stem loop, freeing up residues to base pair with and sequester the palindromic loop of the dimer-promoting DIS hairpin, thereby stabilizing the monomeric form of the RNA. I plan to test this hypothesis using a recently developed nuclear magnetic resonance (NMR) approach that involves nucleotide-specific deuterated RNA prepared by in vitro transcription. I have already developed and published software tools to facilitate the analysis of NMR spectra obtained from large RNAs. I have also obtained NMR spectra of outstanding quality for several differentially labeled, intact HIV-1 5-leader RNAs (371 nucleotides), demonstrating feasibility. Once the secondary structure differences associated with 5-end heterogeneity have been determined, my efforts will focus on determining the three-dimensional structure of the monomeric form of the intact 5-leader. I will test structural elements that appear to be important for regulating RNA splicing, transcriptional activation, and translation by in vivo mutagenesis experiments under the guidance of Dr. Telesnitsky. NMR studies of RNA are challenging: the average size of NMR-derived RNA structures in the RNA Structure Database is only twenty-seven nucleotides, and the largest comprises 155 nucleotides. As such, the proposed NMR studies of the 371-nucleotide 5 leader will push the limits of current NMR technologies. Our findings could lead not only to a more detailed understanding of how HIV RNA structure regulates function, but also to the development of new approaches for the treatment of HIV infection.